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  • 99
    New England Biolabs guanosine triphosphate gtp
    Guanosine Triphosphate Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher guanosine triphosphate gtp
    Guanosine Triphosphate Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher gtp
    The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting <t>TEC</t> ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM <t>GTP</t> in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.
    Gtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TriLink guanosine triphosphate gtp
    The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting <t>TEC</t> ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM <t>GTP</t> in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.
    Guanosine Triphosphate Gtp, supplied by TriLink, used in various techniques. Bioz Stars score: 90/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cytoskeleton Inc guanosine triphosphate gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Valiant guanosine triphosphate gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp, supplied by Valiant, used in various techniques. Bioz Stars score: 91/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Fisher Scientific guanosine triphosphate gtp
    <t>Tubulin-directed</t> activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 <t>P]GTP.</t> GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.
    Guanosine Triphosphate Gtp, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche guanosine triphosphate gtp
    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both <t>ATP</t> and <t>GTP</t> (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P
    Guanosine Triphosphate Gtp, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim guanosine triphosphate gtp
    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both <t>ATP</t> and <t>GTP</t> (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P
    Guanosine Triphosphate Gtp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer guanosine triphosphate gtp biotin 11 gtp
    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both <t>ATP</t> and <t>GTP</t> (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P
    Guanosine Triphosphate Gtp Biotin 11 Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cytoskeleton Inc rho guanosine triphosphate gtp pulldown assay kit
    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both <t>ATP</t> and <t>GTP</t> (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P
    Rho Guanosine Triphosphate Gtp Pulldown Assay Kit, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 89/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore guanosine triphosphate gtp
    (A) Time-dependent release of DOX-D from the <t>ATP-responsive</t> hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM <t>GTP,</t> and (d) untreated.
    Guanosine Triphosphate Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    GE Healthcare guanosine triphosphate gtp binding protein
    (A) Time-dependent release of DOX-D from the <t>ATP-responsive</t> hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM <t>GTP,</t> and (d) untreated.
    Guanosine Triphosphate Gtp Binding Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    List Biological Laboratories guanosine triphosphate gtp binding protein
    (A) Time-dependent release of DOX-D from the <t>ATP-responsive</t> hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM <t>GTP,</t> and (d) untreated.
    Guanosine Triphosphate Gtp Binding Protein, supplied by List Biological Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore gtp
    FL T66W and F138A mutant SaFtsZ proteins have compromised polymerization and GTPase activities. (A) Polymerization of FtsZ proteins at 10 μM was assayed by sedimentation in the presence of <t>GTP</t> and <t>GMPCPP</t> (CPP) with and without the FtsZ inhibitor PC190723 (PC). Pelleted (P) and soluble (S) protein samples were subjected to SDS-PAGE in the same gel lane with a delay. The percentage of pelleted protein was estimated from integration of band intensities. (B) GTPase activity of FtsZ proteins at 10 and 20 μM in the presence of GTP or GMPCPP. (C) Polymerization of FtsZ proteins in the presence of GTP and GMPCPP with and without the FtsZ inhibitor PC190723 (PC) was assessed by negative-stain electron microscopy. All images are at the same magnification (scale bar, 200 nm). WT, wild type.
    Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gtp  (TaKaRa)
    88
    TaKaRa gtp
    FL T66W and F138A mutant SaFtsZ proteins have compromised polymerization and GTPase activities. (A) Polymerization of FtsZ proteins at 10 μM was assayed by sedimentation in the presence of <t>GTP</t> and <t>GMPCPP</t> (CPP) with and without the FtsZ inhibitor PC190723 (PC). Pelleted (P) and soluble (S) protein samples were subjected to SDS-PAGE in the same gel lane with a delay. The percentage of pelleted protein was estimated from integration of band intensities. (B) GTPase activity of FtsZ proteins at 10 and 20 μM in the presence of GTP or GMPCPP. (C) Polymerization of FtsZ proteins in the presence of GTP and GMPCPP with and without the FtsZ inhibitor PC190723 (PC) was assessed by negative-stain electron microscopy. All images are at the same magnification (scale bar, 200 nm). WT, wild type.
    Gtp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cytoskeleton Inc guanosine triphosphate gamma s gtpγs
    FL T66W and F138A mutant SaFtsZ proteins have compromised polymerization and GTPase activities. (A) Polymerization of FtsZ proteins at 10 μM was assayed by sedimentation in the presence of <t>GTP</t> and <t>GMPCPP</t> (CPP) with and without the FtsZ inhibitor PC190723 (PC). Pelleted (P) and soluble (S) protein samples were subjected to SDS-PAGE in the same gel lane with a delay. The percentage of pelleted protein was estimated from integration of band intensities. (B) GTPase activity of FtsZ proteins at 10 and 20 μM in the presence of GTP or GMPCPP. (C) Polymerization of FtsZ proteins in the presence of GTP and GMPCPP with and without the FtsZ inhibitor PC190723 (PC) was assessed by negative-stain electron microscopy. All images are at the same magnification (scale bar, 200 nm). WT, wild type.
    Guanosine Triphosphate Gamma S Gtpγs, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting TEC ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM GTP in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of the OZM at the RNA 3′ end on the kinetics of the incorporation of the next nucleotide ( A ) and the GreA mediated RNA cleavage of the resulting TEC ( B ). (A) TECs were pre-extended by the addition of 10 μM UTP (black), OZM triphosphate (red) or ΨTP (blue) and supplemented with 200 μM GTP in quench flow experiments. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a sum of exponential (corresponds to the fast phase) and stretched exponential (corresponds to the slow phase) functions. The rates and the amplitudes of the fast phase inferred from the data in the graph are presented in the table on the right. The TEC schematic is presented above the graph. (B) TECs were assembled as in (A) and pre-extended with uridine and guanosine or OZM and guanosine by the addition of 10 μM of the corresponding NTPs, gel filtrated and supplemented with 2 μM of GreA. Error bars are ranges of duplicate measurements and the solid lines are the best-fits to a stretched exponential function. The median cleavage times and the fractions of RNA resistant to GreA-mediated cleavage inferred from the data are presented in the table below the graph.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Flow Cytometry

    Eco RNAP, Sce RNAPII and Hsa MT RNAP efficiently incorporate OZM in place of uridine. ( A ) The nucleic acid scaffolds employed in the experiments in (B) and (C). ( B ) Incorporation of OZM in place of uridine: the initial TECs (lanes 1 and 4; top schematics in (A)) were supplemented with UTP and GTP (lanes 2 and 3), OZM triphosphate and GTP (lanes 5 and 6), ΨTP and GTP (lanes 7 and 8). NTPs were added at 20 μM and the reactions were incubated for 2 min at 25°C. RNAs were resolved on 25% urea PAGE. Fractional misincorporations (additional bands) are evident in several lanes. ( C ) OZM triphosphate allows efficient transcription of the sequence position encoding uridine: the initial TEC (lane 1, bottom schematics in (A)) was supplemented with ATP, CTP and GTP (lane 2) and additionally with UTP (lane 3) or OZM triphosphate (lane 4). NTPs were added at 100 μM and the reactions were incubated for 15 s at 25°C. The limited read through the position encoding uridine in the absence of UTP and OZM triphosphate (lane 2) was likely due to the misincorporation of CMP in place of UMP. Each assay was performed in triplicate. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: Eco RNAP, Sce RNAPII and Hsa MT RNAP efficiently incorporate OZM in place of uridine. ( A ) The nucleic acid scaffolds employed in the experiments in (B) and (C). ( B ) Incorporation of OZM in place of uridine: the initial TECs (lanes 1 and 4; top schematics in (A)) were supplemented with UTP and GTP (lanes 2 and 3), OZM triphosphate and GTP (lanes 5 and 6), ΨTP and GTP (lanes 7 and 8). NTPs were added at 20 μM and the reactions were incubated for 2 min at 25°C. RNAs were resolved on 25% urea PAGE. Fractional misincorporations (additional bands) are evident in several lanes. ( C ) OZM triphosphate allows efficient transcription of the sequence position encoding uridine: the initial TEC (lane 1, bottom schematics in (A)) was supplemented with ATP, CTP and GTP (lane 2) and additionally with UTP (lane 3) or OZM triphosphate (lane 4). NTPs were added at 100 μM and the reactions were incubated for 15 s at 25°C. The limited read through the position encoding uridine in the absence of UTP and OZM triphosphate (lane 2) was likely due to the misincorporation of CMP in place of UMP. Each assay was performed in triplicate. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Sequencing

    The effect of OZM on transcription through polythymidine sequences by Eco RNAP. ( A ) TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( B ) Lane profiles from gels in (A) depicting the effect of GreA on the transcription of four- and seven-thymidine tracts. ( C ) TECs arrested at the four-thymidine tract (S326 template) were purified by gel-filtration and supplemented with 100 μM NTPs in the absence and presence of 2μM GreA or GreB for 5 min at 25°C.

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of OZM on transcription through polythymidine sequences by Eco RNAP. ( A ) TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( B ) Lane profiles from gels in (A) depicting the effect of GreA on the transcription of four- and seven-thymidine tracts. ( C ) TECs arrested at the four-thymidine tract (S326 template) were purified by gel-filtration and supplemented with 100 μM NTPs in the absence and presence of 2μM GreA or GreB for 5 min at 25°C.

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Purification, Filtration

    The effect of OZM on processive transcription by Sce RNAPII and Hsa MT RNAP. TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( A ) Transcription through the four-thymidine tract. ( B ) Transcription through the OZM-responsive arrest site. OZM lane traces are shown to the right, the Eco RNAP trace was quantified from the gel in Figure 6B .

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: The effect of OZM on processive transcription by Sce RNAPII and Hsa MT RNAP. TECs were assembled using the scaffolds shown above the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM triphosphate or ΨTP, for 5 min at 25°C. The sequences corresponding to the annealing region of the RNA primer are underlined. Thymidines in the transcribed region are highlighted. Pixel counts were linearly scaled to span the full 8-bit grayscale range within each gel panel. Each assay was performed in triplicate. ( A ) Transcription through the four-thymidine tract. ( B ) Transcription through the OZM-responsive arrest site. OZM lane traces are shown to the right, the Eco RNAP trace was quantified from the gel in Figure 6B .

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques:

    Transcription through a template encoding an OZM-responsive arrest site by Eco RNAP. TECs were assembled using the scaffolds shown to the right of the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM-triphosphate for 5 min at 25°C in the presence or absence of 2 μM GreA. The sequence corresponding to the annealing region of the RNA primer is underlined, thymidines in the transcribed region are highlighted. Each assay was performed in triplicate. ( A ) Transcription through two representative short templates. The lane profiles depict the effect of OZM on transcription of the S245 template (left gel panel). Pixel counts were linearly scaled to span 256 gradations within each gel panel. Gels were pseudocolored using the RGB lookup table shown to the right of the gel panels to visualize the low intensity bands. ( B ) Transcription through the simplified and longer derivative of the S275 template (right gel in A) that encodes the OZM-responsive arrest site. The OZM-responsive arrest site at +7 encodes the main determinants of a consensus pause: pyrimidine at the RNA 3′ end, purine at +1 and Gs at −11 and −10, these determinants are shown in bold. Pixel counts were linearly scaled to span the full 8-bit grayscale range. Lane profiles quantified from the gel are presented on the right. The RNA rescued from the arrest site by GreA in OZM-chase does not quantitatively reappear upstream or downstream of the arrest site (see the text for details and possible explanations).

    Journal: Nucleic Acids Research

    Article Title: Oxazinomycin arrests RNA polymerase at the polythymidine sequences

    doi: 10.1093/nar/gkz782

    Figure Lengend Snippet: Transcription through a template encoding an OZM-responsive arrest site by Eco RNAP. TECs were assembled using the scaffolds shown to the right of the gel panels (only the non-template DNA strands are shown) and chased with 100 μM ATP, CTP, GTP and UTP or OZM-triphosphate for 5 min at 25°C in the presence or absence of 2 μM GreA. The sequence corresponding to the annealing region of the RNA primer is underlined, thymidines in the transcribed region are highlighted. Each assay was performed in triplicate. ( A ) Transcription through two representative short templates. The lane profiles depict the effect of OZM on transcription of the S245 template (left gel panel). Pixel counts were linearly scaled to span 256 gradations within each gel panel. Gels were pseudocolored using the RGB lookup table shown to the right of the gel panels to visualize the low intensity bands. ( B ) Transcription through the simplified and longer derivative of the S275 template (right gel in A) that encodes the OZM-responsive arrest site. The OZM-responsive arrest site at +7 encodes the main determinants of a consensus pause: pyrimidine at the RNA 3′ end, purine at +1 and Gs at −11 and −10, these determinants are shown in bold. Pixel counts were linearly scaled to span the full 8-bit grayscale range. Lane profiles quantified from the gel are presented on the right. The RNA rescued from the arrest site by GreA in OZM-chase does not quantitatively reappear upstream or downstream of the arrest site (see the text for details and possible explanations).

    Article Snippet: GreA facilitated RNA cleavage TEC were prepared by incubating the assembled TEC (1 μM) with 10 μM UTP and GTP or OZM triphosphate and GTP in TB10 for 3 min at 25°C and passed through Zeba™ Spin desalting columns 40K MWCO (Pierce Biotechnology, Rockford, USA) pre-equilibrated with TB0 buffer (40 mM HEPES-KOH pH 7.5, 80 mM KCl, 5% glycerol, 0.1 mM EDTA and 0.1 mM DTT).

    Techniques: Sequencing

    Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Tubulin-directed activities of wt and mutated Op18. (A) Tubulin (5 μM) was incubated with a graded concentration of Op18-wt on ice for 30 min in the presence of [γ- 32 P]GTP. GTP exchange was calculated by determination of tubulin-associated [γ- 32 P]GTP as described in Materials and Methods. (B) [γ- 32 P]GTP was allowed to bind to tubulin in the presence of graded concentrations of Op18 as described for panel A. Unbound [γ- 32 P]GTP was thereafter removed on a desalting column, and [γ- 32 P]GTP-loaded tubulin and Op18 were incubated at 37°C. The mean of duplicate determinations of hydrolyzed GTP, after 40 min of incubation, is shown. (C) Modulation of tubulin GTPase activity by 16 μM concentration of the indicated Op18 derivative was determined as described for panel B. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Incubation, Concentration Assay, Activity Assay

    Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

    doi:

    Figure Lengend Snippet: Modulation of nocodazole-stimulated tubulin GTPase activity by wt and mutated Op18. Tubulin (5 μM) was incubated at 37°C in the presence of [γ- 32 P]GTP in the absence (open bars) or presence (striped bars) of nocodazole (33 μM). The indicated Op18 derivative (16 μM) was also included in the reaction mixtures. The means of duplicate determinations of hydrolyzed GTP, after 120 min of incubation, are shown. Data are representative for at least two independent experiments.

    Article Snippet: The protein concentration of the extracts was adjusted to 1 mg/ml, and the extracts were incubated with GTP (1 mM)–bovine tubulin (10 μM) (Cytoskeleton, Denver, Colo.) in PEM buffer (pH 6.8).

    Techniques: Activity Assay, Incubation

    Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both ATP and GTP (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p acts before Sec22p during homotypic ER fusion. Gluc1 and Gluc2 microsomes were incubated at 27°C in the presence of both ATP and GTP (A) or GTP alone (B). At the indicated times, a portion of the reaction received anti-Sey1p (squares) or anti-Sec22p (triangles) antibodies, or was placed on ice (circles). Luciferase activity was measured after 120 min. Fusion values were normalized to those obtained in reactions that received antibodies or were placed on ice at 120 min. Data represent the means ± SEM (error bars; n = 3). *, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation, Luciferase, Activity Assay

    In vitro homotypic ER fusion requires the atlastin GTPase Sey1p. (A) GTP hydrolysis is required for ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the indicated nucleotide for 90 min. Fusion values were normalized to those obtained using an ATP/GTP-driven reaction incubated at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: In vitro homotypic ER fusion requires the atlastin GTPase Sey1p. (A) GTP hydrolysis is required for ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the indicated nucleotide for 90 min. Fusion values were normalized to those obtained using an ATP/GTP-driven reaction incubated at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Incubation

    Sey1p at its physiological concentration is not sufficient to induce liposome fusion. (A) Proteoliposomes with the indicated Sey1p-to-lipid ratio were generated as described in the Materials and methods. Donor and acceptor proteoliposomes were mixed and incubated at 30°C for 10 min. After GTP and Mg 2+ were added, and NBD fluorescence was measured at 30-s intervals for 30 min. β-Octylglucoside was then added to determine total fluorescence. Fusion is expressed as the percentage of total fluorescence. All experiments were performed multiple times with similar results, and the data shown are representative of all results. (B) Overexpression of Sey1p restores fusion to sec22 Δ microsomes. The indicated microsomes were incubated on ice or at 27°C with GTP/ATP in the absence or presence of anti-Sey1p antibodies for 90 min. SEY1-OE , SEY1 overexpressor. Data represent the means ± SEM (error bars; n = 3). ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p at its physiological concentration is not sufficient to induce liposome fusion. (A) Proteoliposomes with the indicated Sey1p-to-lipid ratio were generated as described in the Materials and methods. Donor and acceptor proteoliposomes were mixed and incubated at 30°C for 10 min. After GTP and Mg 2+ were added, and NBD fluorescence was measured at 30-s intervals for 30 min. β-Octylglucoside was then added to determine total fluorescence. Fusion is expressed as the percentage of total fluorescence. All experiments were performed multiple times with similar results, and the data shown are representative of all results. (B) Overexpression of Sey1p restores fusion to sec22 Δ microsomes. The indicated microsomes were incubated on ice or at 27°C with GTP/ATP in the absence or presence of anti-Sey1p antibodies for 90 min. SEY1-OE , SEY1 overexpressor. Data represent the means ± SEM (error bars; n = 3). ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Concentration Assay, Generated, Incubation, Fluorescence, Over Expression

    ER SNAREs are involved in Sey1p-dependent ER fusion. (A) Sec22p is involved in Sey1p-mediated ER fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups. A Tukey’s test between reactions was performed at 27°C. P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: ER SNAREs are involved in Sey1p-dependent ER fusion. (A) Sec22p is involved in Sey1p-mediated ER fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups. A Tukey’s test between reactions was performed at 27°C. P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation

    In vitro homotypic ER fusion is Rab independent. (A) Homotypic ER fusion is insensitive to Rab inhibition by Gdi1p/Gyp1p. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C with ATP/GTP in the absence or presence of his 6 -Gdi1p and his 6 -Gyp1p for 90 min. For 1×Gdi1p/Gyp1p, the concentrations of his 6 -Gdi1p and his 6 -Gyp1p were 1.5 and 2.5 µM, respectively; the corresponding concentrations for 4×Gdi1p/Gyp1p were 6 and 10 µM, respectively. Data represent the means ± SEM (error bars; n = 3). ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: In vitro homotypic ER fusion is Rab independent. (A) Homotypic ER fusion is insensitive to Rab inhibition by Gdi1p/Gyp1p. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C with ATP/GTP in the absence or presence of his 6 -Gdi1p and his 6 -Gyp1p for 90 min. For 1×Gdi1p/Gyp1p, the concentrations of his 6 -Gdi1p and his 6 -Gyp1p were 1.5 and 2.5 µM, respectively; the corresponding concentrations for 4×Gdi1p/Gyp1p were 6 and 10 µM, respectively. Data represent the means ± SEM (error bars; n = 3). ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Inhibition, Incubation

    The SNARE chaperones Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion. (A) Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion, but not in GTP-driven fusion. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Some reactions were treated with anti-Sec17p antibodies, anti-Sec18p antibodies, or recombinant his 6 -Sec18p at the indicated concentrations. Data represent the means ± SEM (error bars; n = 3). **, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: The SNARE chaperones Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion. (A) Sec17p and Sec18p are involved in ATP/GTP-driven ER fusion, but not in GTP-driven fusion. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of GTP or ATP/GTP for 90 min. Some reactions were treated with anti-Sec17p antibodies, anti-Sec18p antibodies, or recombinant his 6 -Sec18p at the indicated concentrations. Data represent the means ± SEM (error bars; n = 3). **, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Incubation, Recombinant

    Development of an in vitro homotypic ER fusion assay. (A) Schematic representation of chimeric proteins for the Gluc PCA. (B) Assay scheme; see Results for details. Microsomes isolated from BJ-Gluc1 yeast cells overexpressing ssZIP-Gluc1-HDEL under the control of an ADH1 promoter (Gluc1 microsomes) were mixed with microsomes isolated from BJ-Gluc2 yeast cells overexpressing ssZIP-Gluc2-HDEL (Gluc2 microsomes) and then incubated at 27°C in the presence of GTP and ATP. After 90 min, the luciferase substrate coelenterazine was added, and luciferase activity was measured. Excess GST-ZIP was added to block extra-luminal reconstitution of functional Gluc caused by membrane destabilization or rupture during incubation. (C) Expression of Gluc PCA fragments in isolated microsomes. The expression of Gluc PCA fragments was analyzed by immunoblotting using the indicated antibodies. Kar2p, an ER-resident protein, was used as a loading control. (D) GTP-driven homotypic ER fusion is markedly enhanced by ATP. Gluc1 and Gluc2 microsomes were mixed and incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent means ± SEM (error bars; n = 3). RLU, relative luminescence unit. ***, P

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Development of an in vitro homotypic ER fusion assay. (A) Schematic representation of chimeric proteins for the Gluc PCA. (B) Assay scheme; see Results for details. Microsomes isolated from BJ-Gluc1 yeast cells overexpressing ssZIP-Gluc1-HDEL under the control of an ADH1 promoter (Gluc1 microsomes) were mixed with microsomes isolated from BJ-Gluc2 yeast cells overexpressing ssZIP-Gluc2-HDEL (Gluc2 microsomes) and then incubated at 27°C in the presence of GTP and ATP. After 90 min, the luciferase substrate coelenterazine was added, and luciferase activity was measured. Excess GST-ZIP was added to block extra-luminal reconstitution of functional Gluc caused by membrane destabilization or rupture during incubation. (C) Expression of Gluc PCA fragments in isolated microsomes. The expression of Gluc PCA fragments was analyzed by immunoblotting using the indicated antibodies. Kar2p, an ER-resident protein, was used as a loading control. (D) GTP-driven homotypic ER fusion is markedly enhanced by ATP. Gluc1 and Gluc2 microsomes were mixed and incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent means ± SEM (error bars; n = 3). RLU, relative luminescence unit. ***, P

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: In Vitro, Single Vesicle Fusion Assay, Isolation, Incubation, Luciferase, Activity Assay, Blocking Assay, Functional Assay, Expressing

    Sey1p physically interacts with ER SNAREs. (A and B) Sey1p physically interacts with the ER SNARE proteins Sec22p and Ufe1p. Microsomes isolated from BJ3505 were detergent-solubilized and incubated with anti-Sec22p antibodies (A), anti-Ufe1p antibodies (B), or preimmune IgG (control) in the presence of protein A Sepharose. Protein A Sepharose–bound material was then analyzed by immunoblotting using the indicated antibodies. (C) Sec18p regulates the interaction between Sec22p and Sey1p. BJ3505 microsomes were preincubated in the absence or presence of anti-Sec18p antibodies for 10 min at 4°C. After ATP/GTP was added, microsomes were further incubated for 40 min. During incubation, some samples received anti-Sec18p antibodies at the indicated time points. Sec22p was precipitated using anti-Sec22p antibody–conjugated Dynabeads, and bound proteins were analyzed by immunoblotting using the indicated antibodies. (D) Detection of an in vivo interaction between Sec22p and Sey1p by DSP cross-linking. BJ3505 spheroplasts were incubated in the absence or presence of 4 mM DSP for 30 min at 4°C. After DSP was quenched, detergent-solubilized spheroplasts were subjected to immunoprecipitation using anti-Sec22p antibodies or preimmune IgG (preIgG). Cross-links were cleaved using β-mercaptoethanol in SDS sample buffer, and proteins cross-linked with Sec22p were analyzed by immunoblotting using the indicated antibodies. The asterisk indicates nonspecific bands. All experiments were performed multiple times with similar results, and the data shown are representative of all results.

    Journal: The Journal of Cell Biology

    Article Title: SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

    doi: 10.1083/jcb.201501043

    Figure Lengend Snippet: Sey1p physically interacts with ER SNAREs. (A and B) Sey1p physically interacts with the ER SNARE proteins Sec22p and Ufe1p. Microsomes isolated from BJ3505 were detergent-solubilized and incubated with anti-Sec22p antibodies (A), anti-Ufe1p antibodies (B), or preimmune IgG (control) in the presence of protein A Sepharose. Protein A Sepharose–bound material was then analyzed by immunoblotting using the indicated antibodies. (C) Sec18p regulates the interaction between Sec22p and Sey1p. BJ3505 microsomes were preincubated in the absence or presence of anti-Sec18p antibodies for 10 min at 4°C. After ATP/GTP was added, microsomes were further incubated for 40 min. During incubation, some samples received anti-Sec18p antibodies at the indicated time points. Sec22p was precipitated using anti-Sec22p antibody–conjugated Dynabeads, and bound proteins were analyzed by immunoblotting using the indicated antibodies. (D) Detection of an in vivo interaction between Sec22p and Sey1p by DSP cross-linking. BJ3505 spheroplasts were incubated in the absence or presence of 4 mM DSP for 30 min at 4°C. After DSP was quenched, detergent-solubilized spheroplasts were subjected to immunoprecipitation using anti-Sec22p antibodies or preimmune IgG (preIgG). Cross-links were cleaved using β-mercaptoethanol in SDS sample buffer, and proteins cross-linked with Sec22p were analyzed by immunoblotting using the indicated antibodies. The asterisk indicates nonspecific bands. All experiments were performed multiple times with similar results, and the data shown are representative of all results.

    Article Snippet: In vitro ER membrane fusion assay The standard ER fusion reaction (50 µl) contained 5 µg of Gluc1 microsomes, 5 µg of Gluc2 microsomes, reaction buffer (10 mM Pipes-KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2 , and 200 mM sorbitol), an energy-regenerating system (1 mM MgCl2 , 1 mg/ml creatine kinase, and 29 mM creatine phosphate), 264 nM Pbi2p (IB2 ), 10 µM coenzyme A, 1 mM ATP and/or 1 mM GTP (Roche), and 100 µM GST-ZIP.

    Techniques: Isolation, Incubation, In Vivo, Immunoprecipitation

    (A) Time-dependent release of DOX-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM GTP, and (d) untreated.

    Journal: Chemical Science

    Article Title: pH- and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules †Electronic supplementary information (ESI) available: Nucleic acid sequences; characterization of nucleic acid-modified copolymers; SEM images, confocal images of microcapsules; calibration curves of TMR-D, TR-D, and DOX-D; synthesis of DOX-D; CD spectra of pH-responsive hydrogel; characterization of hydrogel membranes. See DOI: 10.1039/c6sc04770jClick here for additional data file.

    doi: 10.1039/c6sc04770j

    Figure Lengend Snippet: (A) Time-dependent release of DOX-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). The released DOX-D was acidified to enable the cleavage of fluorescent doxorubicin from dextran. (B) Fluorescence spectra corresponding to the release of doxorubicin upon exposing the ATP-responsive hydrogel microcapsules to different concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, (f) 50, and (g) 75 mM. Inset: derived calibration curve of the fluorescence intensities of the released doxorubicin at λ em = 600 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of doxorubicin from the ATP-responsive microcapsules loaded with doxorubicin-dextran and exposed to different nucleoside triphosphates for a fixed time interval of 30 minutes: (a) 25 mM ATP, (b) 25 mM CTP, (c) 25 mM GTP, and (d) untreated.

    Article Snippet: Reagents and materials Magnesium chloride, sodium chloride, doxorubicin hydrochloride (DOX), 4-carboxyphenylboronic acid, dextran (MW = 40 kDa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), N -(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDC), N -hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (MW = 30, 450, and 1250 kDa), 2-(N -morpholino)ethanesulfonic acid (MES), ammonium persulfate (APS), N ,N ,N ′,N ′-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kDa), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Derivative Assay

    (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Journal: Chemical Science

    Article Title: pH- and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules †Electronic supplementary information (ESI) available: Nucleic acid sequences; characterization of nucleic acid-modified copolymers; SEM images, confocal images of microcapsules; calibration curves of TMR-D, TR-D, and DOX-D; synthesis of DOX-D; CD spectra of pH-responsive hydrogel; characterization of hydrogel membranes. See DOI: 10.1039/c6sc04770jClick here for additional data file.

    doi: 10.1039/c6sc04770j

    Figure Lengend Snippet: (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Article Snippet: Reagents and materials Magnesium chloride, sodium chloride, doxorubicin hydrochloride (DOX), 4-carboxyphenylboronic acid, dextran (MW = 40 kDa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), N -(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDC), N -hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (MW = 30, 450, and 1250 kDa), 2-(N -morpholino)ethanesulfonic acid (MES), ammonium persulfate (APS), N ,N ,N ′,N ′-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kDa), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Derivative Assay

    FL T66W and F138A mutant SaFtsZ proteins have compromised polymerization and GTPase activities. (A) Polymerization of FtsZ proteins at 10 μM was assayed by sedimentation in the presence of GTP and GMPCPP (CPP) with and without the FtsZ inhibitor PC190723 (PC). Pelleted (P) and soluble (S) protein samples were subjected to SDS-PAGE in the same gel lane with a delay. The percentage of pelleted protein was estimated from integration of band intensities. (B) GTPase activity of FtsZ proteins at 10 and 20 μM in the presence of GTP or GMPCPP. (C) Polymerization of FtsZ proteins in the presence of GTP and GMPCPP with and without the FtsZ inhibitor PC190723 (PC) was assessed by negative-stain electron microscopy. All images are at the same magnification (scale bar, 200 nm). WT, wild type.

    Journal: mBio

    Article Title: A Polymerization-Associated Structural Switch in FtsZ That Enables Treadmilling of Model Filaments

    doi: 10.1128/mBio.00254-17

    Figure Lengend Snippet: FL T66W and F138A mutant SaFtsZ proteins have compromised polymerization and GTPase activities. (A) Polymerization of FtsZ proteins at 10 μM was assayed by sedimentation in the presence of GTP and GMPCPP (CPP) with and without the FtsZ inhibitor PC190723 (PC). Pelleted (P) and soluble (S) protein samples were subjected to SDS-PAGE in the same gel lane with a delay. The percentage of pelleted protein was estimated from integration of band intensities. (B) GTPase activity of FtsZ proteins at 10 and 20 μM in the presence of GTP or GMPCPP. (C) Polymerization of FtsZ proteins in the presence of GTP and GMPCPP with and without the FtsZ inhibitor PC190723 (PC) was assessed by negative-stain electron microscopy. All images are at the same magnification (scale bar, 200 nm). WT, wild type.

    Article Snippet: GTP (Sigma, St. Louis, MI), and GMPCPP (Jena Bioscience, Jena, Germany) hydrolysis by FL SaFtsZ proteins was monitored by detecting the release of inorganic phosphate with the malachite green assay ( ) in samples containing 10 or 20 μM protein in buffer HKM at 25°C.

    Techniques: Mutagenesis, Sedimentation, Conditioned Place Preference, SDS Page, Activity Assay, Staining, Electron Microscopy